In the early 1980's several groups isolated and characterized growth hormone releasing factor (GRF).
GRF (also called Somatorelin) is a peptide secreted by the hypothalamus, which acts on its receptor and can promote the release of growth hormone (GH) from the anterior pituitary. It exists as 44-, 40-, or 37-amino acid peptide; the 44-amino acid form may be converted physiologically into shorter forms. All three forms are reported to be active, the activity residing mainly in the first 29 amino acid residues. A synthetic peptide corresponding to the 1-29 amino acid sequence of human GRF [GRF(1-29)], also called Sermorelin, has been prepared by recombinant DNA technology as described in European Patent EP 105 759.
Sermorelin has been used in the form of acetate for the diagnosis and treatment of growth hormone deficiency.
GRF has indeed a therapeutic value for the treatment of certain growth hormone related disorders. The use of GRF to stimulate the release of GH is a physiological method in promoting long bone growth or protein anabolism.
One problem associated with the use of GRF relates to its short biological half-life (about 12 to 30 minutes). The GRF(1-29)-NH2 is subject to enzymatic degradation and is rapidly degraded in the plasma via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala2 and Asp3.
It is therefore advantageous to develop biologically stable, long acting GRF analogues using specific chemical modification of GRF, in order to prevent or slow down enzymatic degradation.
Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic polymer of general formula H(OCH2CH2)nOH, wherein n≧4. Its molecular weight could vary from 200 to 20,000 Dalton.
It has been demonstrated that the chemical conjugation of PEG in its mono-methoxylated form to proteins and/or peptides significantly increases their duration of biological action. Like carbohydrate moieties in a glycoprotein, PEG provides a protective coating and increases the size of the molecule, thus reducing its metabolic degradation and its renal clearance rate.
PEG conjugation is an already established methodology for peptide and protein deivery pioneered by the fundamental studies of Davis and Abuchowski (Abuchowski et al., 1977a and 1977b). PEG conjugation to peptides or proteins generally resulted in non-specific chemical attachment of PEG to more than one amino acid residue. One of the key issues with this technology is therefore finding appropriate chemical methods to covalently conjugate PEG molecule(s) to specific amino acid residues.
Various PEG-protein conjugates were found to be protected from proteolysis and/or to have a reduced immunogenicity (Monfardini et al., 1995; and Yamsuki et al., 1988).
One approach was recently proposed for the site-specific conjugation of PEG to low molecular weight peptides, such as GRF, which was prepared by solid-phase peptide synthesis. In these conjugates a pegylated amino acid, prepared in advance, was introduced into the peptide sequence during the solid-phase synthesis. This procedure, however, dramatically complicates product purification that is known to be the critical step in solid phase synthesis. The presence of PEG, for its high molecular weight and its polydispersivity, is likely to yield final products with unacceptable impurities and/or products with missing amino acids, the latter being considered to occur commonly in the Merrifield procedure.
Mono-pegylation, meaning that only one PEG molecule is attached, using solid-phase synthesis to specific amino acid residues of [Ala15]-GRF(1-29)-NH2 has been recently reported in the literature (Felix et al., 1995). This study shows that [Ala15]-GRF(1-29)-NH2 pegylated at residues 21 or 25 retains the fill in-vitro potency of the parent [Ala15]-GRF(1-29)-NH2. There is however no in-vivo data to show whether these pegylated conjugates exhibit a longer duration of action with respect to the non-pegylated counterpart.
More recently, it has been demonstrated (Campbell et al., 1997) that the attachment of PEG with different molecular weights to the C-terminus of several analogs of GRF, again using solid-phase synthesis, had enhanced duration of action in both pig and mouse models as compared to the non-pegylated counterpart.
EP 400 472 and EP 473 084 disclose PEG derivatives obtained by using specific activated PEG derivatives. In the Examples also GRF is made to react with such activated PEG derivatives, however the specific GRF-PEG which are obtained according to such patent applications are not exactly identified nor in any way characterized. There is no mention of a specific PEG attachment site.
WO 99/27897 discloses a method for the site-specific preparation of GRF-PEG conjugates containing one or more than one PEG units (per GRF) covalently bound to Lys12 and/or Lys21 and/or Nα, characterized in that the conjugation reaction between the GRF peptide and activated PEG is carried out in solution and the desired GRF-PEG conjugate is purified by chromatographic methods. According to such patent application the solvent was selected from the group consisting of a highly concentrated nicotinamide aqueous solution, a buffered aqueous solution of a defolding agent (such as urea) or a polar organic solvent selected among dimethyl sulfoxide, dimethyl formamide/buffer or acetonitrile/buffer. Dimethyl sulfoxide (DMSO) was mainly used in the Examples.